MICROBIOLOGY 1: BACTERIOLOGY

BIOCHEMICAL TEST

INTRODUCTION

The bacteria thus isolated needs to be further identified to genus and species level. The identification is
required so as to cure the illness or the infection caused due to the bacteria by using appropriate antibiotics.
Identification also holds significance for epidemiological purposes

OBJECTIVES

State the specific diagnostic purpose of each test methodology

Briefly describe the test principles associated with each test methodology

DEFINITION OF TERMS

 Alpha hemolysis – partial or “green” hemolysis associated with reduction of red cell hemoglobin. Alpha
hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green
methemoglobin. It exhibit incomplete haemolysis with 1-2 mm wide
 Beta hemolysis – is a complete lysis of red cells in the media around and under the colonies: the area
appears lightened (yellow) and transparent.
 Gamma hemolysis – lack of hemolysis in the area around a bacterial colony.
 Inoculum - The bacteria that are transferred during inoculation. The inoculum, it is hoped, consists only of
the desired species of bacteria and does not include any contaminants
 Zone of Inhibition - This is an area of media where bacteria are unable to grow, due to presence of a drug
that impedes their growth.
 Deamination – removal of the amine group from a molecule
 Decarboxylation – removal of the carbon dioxide from a molecule

BIOCHEMICAL TESTS:

The staining is followed by use of various biochemical reagents and tests to get closer to the identification of
bacteria. There are many biochemical tests available for bacterial identification. Few of them are required to
be carried out depending upon the bacteria. The commonly used biochemical tests are as mentioned below:

MOTILITY TESTING
 Purpose:
o These test are used to determine whether an enteric organism is motile. An organism must have
flagella to be motile
 Principle :
o The inoculum is stabbed into the center of a semi -solid agar deep.
o Bacterial motility is evident by a diffuse zone of growth extending out from the line of
inoculation.
o Some organisms grow throughout the medium, whereas others show small areas or nodules that
grow out from the line of inoculation

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 Method :
o Touch a straight needle to a colony of a young (18-24hour) culture growing on agar medium
o Stab once to a depth of only 1/3 to ½ inch on the midle of the tube
o Incubate at 35-37C and examine daily for up to 7 days.
 RESULTS:
o POSITIVE: Motile organism will spread out into the medi um from the site of inoculation
o NEGATIVE: Nonmotile organisms remain at the site of inoculation

 Characteristics motility of some organisms:

o Darting Motility – Campylobacter sp
o Tumbling end-over-end motility – Listeria monocytogenes
o Gliding motility – Capnocytophagia sp
o Swarming motility – Proteus sp
MICRODASE OR MODIFIED OXIDASE TEST

 Purpose:
o This test used to differentiate gram positive, catalase positive cocci
 Principle:
o The microdase test is a rapid method to differentiate Staphylococcus from Micrococcus spp by
detection of the enzyme oxidase.
o In the presence of atmospheric oxygen, the oxidase enzyme reacts with oxidase reagent and
cytochrome C to form the colored compound, indophenol
 Method:
o Using a wooden applicator stick, rub a small amount of several colonies of an 18 to 24 hour pure
culture grown on blood agar onto a small area of the microdase disk.
o Incubate at room temperature for 2 minutes
o Reagent: 6% Tetramethyl paraphenylene diamine dihydrochloride with dimethyl sulfoxide
o Positive result: Dark Blue
 {+} Micrococcus luteus
 {-} Staphylococcus

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CATALASE TEST

 Purpose:
o To differentiates catalase positive microorganism and staphylococcal species from Catalase
Negative Streptococcal species
 Principle:
o Aerobic and facultative anaerobic organism produce two toxins during normal metabolism,
hydrogen peroxide and superoxide radical.
o These bacteria have two enzyme that detoxify the products of normal metabolism. One of these
enzymes, catalase, is capable of converting hydrogen peroxide to water and oxygen.
o The presence of enzyme in inoculum is evidenced when a small inoculum introduced to hydrogen
peroxide, causes rapid elaboration of oxygen bubbles.
o The lack of catalase is evident by lack or weak bubble production
 Method:
o Use a loop or sterile wooden stick to transfer small amount of colony growth to the surface of a
clean, dry glass slide.
o Place a drop of 3% hydrogen peroxide on the colony
o Observe production of oxygen bubbles.
o Positive result: Gas production, Bubbles
{+} Staphylococcus and Micrococcus
{-} Streptococcus

COAGULASE TEST

 Purpose:
o To differentiate Staphylococcus aureus from Coagulase Negative Staphylococci
 Principle:
o Staphylococcus aureus produces two forms of coagulase bound and free.
o BOUND COAGULASE: clumping factor is bound to the cell wall and reacts directly with
fibrinogen. This results in precipitation of fibrinogen on the staphylococcal cell, causing the cells
to clump when a bacterial suspension is mixed with plasma.
o FREE COAGULASE: an extracellular protein enzyme that causes the formation of clot when S.
aureus incubated with plasma. The clotting mechanis m involves activation of plasma coagulase
reacting factor (CRF) which is a modified or derived thrombin molecule, to form coagulase-CRF
complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.
 METHOD:
o SLIDE TEST
 Place a drop of coagulase plasma on a clean dry glass slide
 Place a drop of distilled water or saline next to the of a plasma as a control

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 With a loop, emulsify a portion of the isolated colony being tested in each drop, inoculating the
water or saline first. Try to create smooth suspension
 Mix well with a wooden applicator stick
 Rock the slide gently for 5-10 sec.
 Expected results:
 POSITIVE {+} = macroscopic clumping on 10 sec or less on coagulated plasma drop and
no clumping in saline or water
 NEGATIVE {-} = No clumping on either drop
o TUBE TEST
 Emulsify several colonies in 0.5ml of rabbit plasma to give milky suspension.
 Incubate tube at 35-37C in ambient air for 4 hours
 Check for clot formation
 POSITIVE(+) : Clot of any size
 NEGATIVE(-) : NO clot formation

NOVOBIOCIN SUSCEPTIBILITY TESTING

 Purpose:
o Differentiates the coagulase-negative Staphylococcus saprophyticus from Staphylococcus
epidermidis
o Reagent: 5ug of novobiocin and sheep blood agar
o Positive result: zone of inhibition greater than 16 mm
 {+} susceptible: Staphylococcus epidermidis
 {-} resistant : Staphylococcus saprophyticus

BACITRACIN SUSCEPTIBILITY TESTING

 Purpose:
o This test used for presumptive identification and differentiation of beta-hemolytic group A
Streptococci (Streptococcus pyogenes) from other beta hemolytic streptococci.
o It also used to distinguish Staphylococcus species from Micrococci

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o Reagent: 0.04U bacitracin disk(TAXO A)

 Principle:
o The antibiotic bacitracin inhibits the synthesis of bacterial cell walls.
o A disk(Taxo A) impregnated with a small amount of bacitracin (0.04 units) is placed on agar plate,
allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms.
 METHOD
o Using inoculating loop, streak two or three suspect colonies of a pure culture onto blood agar plate
o Using heated forceps, place bacitracin on the 1 st quadrant
o Incubate the plate for 18-24 hours at 35-37C for Staphylococci and 5-10% CO2 for Streptococci
o Look for zone of inhibition.
o EXPECTED RESULTS
 {+} susceptible: Any zone of inhibition greater than 10mm
 Streptococcus pyogenes, Micrococcus, Stomatococcus
 {-} resistant: No zone of inhibition
 Streptococcus agalactiae, Staphylococcus aureus

OPTOCHIN SUSCEPTIBILITY TESTING

 Purpose:
o To determine the effect of optochin(ethyl hydrocupreine hydrochloride) on an organism.
Optochin lyses pneumococci {Streptococcus pneumoniae} (sensitive), but alpha -
streptococci{Viridans Streptococci} are resistant
 PRINCIPLE:
o Optochin is an antibiotic that interferes with the ATPase and production of adenosine
triophosphate(ATP) in microorganism.
o The optochin impregnated disk (Taxo P) is placed on a lawn of organism on a sheep blood agar
plate, allowing the antibiotic to diffuse into the medium.
o The antibiotic inhibits the growth of susceptible organism, creating a clearing, or zone of
inhibition, around the disk.

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 METHOD:
1.Using inoculating loop, streak two or three suspect colonies of pure culture onto half of a 5%
sheep blood agar plate.
2. Place an optochin disk in the 1 st quadrant of streak.
3. Incubate the plate for 18-24 hours at 35C in 5% CO2.
4. Measure the zone of inhibition
RESULT:
Zone of Inhibition
 [ + ] ≥14 mm in diameter : Streptococcus pneumoniae
 [ - ] No zone of inhibition: Viridans Streptococci

BILE SOLUBILITY TEST

 Purpose:
o Test to differentiate Streptococcus pneumoniae from alpha hemolytic streptococci
 Principle:
o Bile or a solution of bile salt(e.g sodium desoxycholate) rapidly lyses pneumonococcal colonies
o Lysis depends on the presence of an intracellular autolytic enzyme, amidase.
o Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus
accelerating the organism’s natural autolytic process.
 Method:
o After 12 to 24 hours incubation on blood agar plate, place 1 to 2 drops of 10% sodium
desoxycholate on well isolated colony.
o Gently wash liquid over the colony without dislodging the colony from the agar
o Incubate the plate at 35-37C in ambient air for 30 mins
o Examine for lysis of colony
o RESULTS:
 POSITIVE = Colony disintegrates, an imprint of the lysed colony may remain on the zone
 Streptococcus pneumoniae (bile soluble)
 NEGATIVE = intact colonies
 Enterococcus faecalis(bile insoluble)

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Bile Esculin Test

 Purpose:
o This test is used for the presumptive identification of Enterococci and organisms in the Streptococcus
bovis group
o The test differentiates enterococci and group-D streptococci from non group D viridans streptococci
 Principle:
o Gram positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in
this medium
o Organism capable of growth in the presence of 4% bile and able to hydrolize esculin to esculetin.
Esculetin reacts with Fe3+ and forms a dark brown to black precipitate
 Method:
o Inoculate one to two colonies from an 18 to 24 hour culture onto the surface of the slant
o Incubate at 35-37C in ambient air for 48 hours.
o Results:
(+) = growth and blackening of the agar slants
Enterococcus faecalis
(-) = growth and no blackening of agar slant
= No growth, no color change
Streptococcus pyogenes

Salt Tolerance Test

 Purpose:
o This test is used to determine the ability of an organism to grow in high concentrations of salt.
Used to differentiate enterococci from non-enterococci
 Principle:
o The salt tolerance test is a selective and differential medium.
o Enterococci are resistant to high salt concentration. A heart infusion broth containing 6.5% NaCl
is used as the test medium
o The broth contains a small amount of glucose and bromcresol purple as the indicator for acid
production.
 Method:
o Inoculate one or two colonies from an 18- to 24-hour culture into 6.5%NaCl broth.
o Incubate the tube at 35-37C in ambient air for 48 hours.
o Check daily for growth
o Expected Results:
[+] = visible turbidity in the broth, with or without

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color change from purple to yellow

[-} = No turbidity and no color change

CAMP Test (Christie, Atkins and Munch Peterson)

 Purpose:
o Differentiate group B (Streptococcus agalactiae) from other Streptococcal species. Listeria
monocytogenes also produces positive for CAMP reaction
 Principle:
o Certain organisms (including group B streptococci) produce a diffusible extracellular hemolytic
protein(CAMP factor) that acts synergistically with the beta -lysin of Staphylococcus aureus to cause
enhance lysis of red blood cells
o Group B streptococci are streaked perpendicular to streak of S. aureus
 Method:
1.Streak a beta lyin producing strain of S. aureus down the center of a sheep blood agar plate
2. Streak organisms across the plate perpendicular to the S. aureus within 2 mm
3. Incubate overnight at 35-37C at ambient room
o Result:
(+) = enhance hemolysis is indicated by an arrowhead-shaped zone of beta-hemolysis at the
juncture of the two organisms
Streptococcus agalactiae
(-) = No enhancement of hemolysis
Streptococcus pyogenes

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Leucine Aminopeptide (LAP) Test

 Purpose:
o Test used for the presumptive identification of Catalase- negative gram-positive cocci
 Principle:
o The LAP disk is a rapid test for the detection of the enzyme leucine aminopeptidase.
o Leucine-beta-napthylamide-impregnated disks serve as a substrate for the detection of leucine
aminopeptidase
o After hydrolysis of the substrate by the enzyme, the resulting beta -naphthylamine produces a
red color upon addition of cinnamaldehyde reagent
 Method:
o Before inocubation, slightly dampen the LAP disk with reagent grade water.
o Using a wooden applicator stick, rub a small amount of several colonies of an 18 - to 24-hour
pure culture onto a small area of the LAP disk.
o Incubate at RT for 5 mins
o After incubation, add 1 drop of cinnamaldehyde
o Results:
(+) Red color within 1 minute after adding of cinnamaldehyde reagent
Enterococcus faecalis
(-) No color Change
Aerococcus viridans

INDOLE TEST

 Purpose:
o This test is used to identify organism that produce the enzyme tryptophanase
 Principle:
o Bacteria with tryptophanase is capable of hydrol yzing tryptophan to pyruvate, ammonia and
indole detected by its ability to combine with certain aldehydes to form a colored compound
 Three (3) methods of Indole Detection:
 Kovac’s
 Addition of para-dimethylaminobenzaldehyde to the sample will yield a red ring as positive
reaction.

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2. Ehrlich’s
 same chemicals as the Kovac preparation but it also contains absolute ethyl alcohol, making it
flammable
 more sensitive of detecting small amounts of indole.

3. Spot Indole Test

 Rapid detection
 Easy visualization of blue-green colored compound as indole reacts with cinnamaldehyde
 No change in color or slightly pink is negative reaction

OXIDASE TEST

 Purpose:
o Determines the presence of cytochrome oxidase activity in microorganisms for identification of
oxidase-negative Enterobacteriaceae, differentiating them from other gram-negative bacilli
 Principle:

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o To determine the presence of bacterial cytochrome oxidase using the oxidation of substrate
tetramethyl-p-phenylenediamine dihydrochloride to indophenol, a dark purple colored end
product.
o No color development indicates a negative test and the absence of the enzyme
o Expected Results:
[ + ] = dark purple color within 10 seconds
Pseudomonas
[ - ] = absence of color

Methyl-Red/Voges-Proskauer(MRVP test)

 Purpose:
o The combination test of MR and VP differentiates members of the Enterobacteriaceae
 Principle:
 Methyl Red
 Detects mixed acid fermentation that lowes the pH of the broth
 MR indicator is added after incubation, MR red (+) at pH 4.4 and yellow (-) at pH 6.2
 Voges-Proskauer
 detects the organism’s ability to convert the acid products to acetoin and 2,3 -butanediol.
 Organisms capable of using VP pathway produce a smaller amount of acid during glucose
fermentation and therefore do no produce a color change when the MR indicator is added
 Secondary reagent is added, alpha -napthol, followed by potassium hydroxide (KOH), a positive
test is indicated by a red color complex
 Results:
MR (+) and VP (-) = Escherichia coli
MR(-) and VP(-) = Enterobacter aerogenes

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CITRATE UTILIZATION

 Purpose:
o To identify organisms capable of using sodium citrate as the sole carbon source and inorganic
ammonium salts as the sole nitrogen source
o This test is part of IMViC(indole, methyl red, Voges -Proskauer and Citrate), which is used to
differentiate Enterobacteriaceae from other gram negative rods.
 Principle:
o Bacteria produce an enzyme, citrate-permease, capable of converting citrate to pyruvate for energy
production.
o Bacteria capable of growth in the medium use the citrate and convert ammonium phosphate to
ammonia and ammonium hydroxide creating an alkaline pH turning the bromthymol blue indicator
from Green to Blue(+)
Results:

[+] = growth on the medium and Blue color

Enterobacter aerogenes
[-] = absence of growth
Escherichia coli

UREASE TEST (Christensen’s Method)

 Purpose:
o This test is used to determine organism’s ability to produce the enzyme urease, which hydrolizes
urea. Proteus sp. may be presumptively identified by the ability to rapidly hydrolize urea
 Principle:
o Urea is the product of decarboxylation of amino acids
o Hydrolysis of urea produces ammonia and CO2
o Ammonia alkalinizes the medium and pH shift is detected by the color change of phenol red from
light orange at pH 6.8 to magenta (pink) at pH 8.1
 Results:
[+] = change in color of slant from light orange to magenta(24hrs)
Proteus vulgaris
*Weak positive = several days becomes +
Klebsiella pneumoniae
[-] = No color change
Escherichia coli

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Ortho-nitrophenyl-beta-D-Galactopyranoside (ONPG) Test

 Principle:
o ONPG enters the cells of organism that do not produce permease but are capable of hydrolyzing
the ONPG to galactose and a yellow compound,o-nitrophenol, indication the presence of beta -
galactosidase
 Results:
[+] = Yellow
= Presence of beta-galactosidase
Shigella sonnei
[-] = Colorless
= Absence of enzyme
Salmonella typhimurium
Phenylalanine Deaminase (PAD) Test

 Principle:
o Microorganisms that produce phenylalanine Deaminase remove amin (NH2) from phenylalanine
resulting in the production of ammonia (NH3) and phenylpyruvic acid.
o Phenylpyruvic acid is detected by adding a few drops of 10% ferric chloride producing a green -
colored complex
 Results:
[+] = Green color develops on slant after addition of ferric chloride
Proteus mirabilis
[-] = Slant retains original color after addition of ferric chloride
Escherichia coli

L-Pyrolidonyl Arylamidase(PYR) Test

 Purpose:
o Presumptive identification of Group A streptococci( Streptococcus pyogenes) and enterococci by
the presence of the L-pyrrolidonyl anylamidase

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 Principle:
o The enyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl-B-naphthylamide substrate
to produce a B-naphthulamine
o B-naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde reagent
by the production of a bright red precipitate
 Results:
[+] = bright red color within 5 minutes
Enterococcus faecalis
[-] = No color change orange color
Escherichia coli

Hippurate Hydrolysis

 Purpose:
o Production of the enzyme hippuricase is used for the presumptive identification of a variety of
microorganism
o Determine if an organism can hydrolyze Na Hippurate to Benzoic Acid and glycine
 Principle:
o End product of hydrolysis of hippuric acid by hippuricase include glycine and benzoic acid
o Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during process
o The end products of the ninhydrin oxidation react to form purple-colored products.
 Results:
[+] = deep purple color
Streptococcus agalactiae (Group B)
[-] = colorless or slightly yellow pink
Streptococcus pyogenes (Group A)

X and V Factor

 Purpose:
o Used to differentiate Haemophilus species
o Members of the genus haemophilus require accessory growth factors in vitro. Some
Haemophilus spp require X factor(hemin) alone, V factor(nicotinamide adenine dinycleotide-
NAD) alone, or a combination of two.
 Principle:
o A lawn of the test organism is streaked onto TSA, NA, heart infusion agar or Haemophilus agar

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o The impregnated disks or strips(X, V, XV) placed directly on the confluent inoculation, allowing
diffusion of the accessory growth factor into the medium
o The organisms will grow only around the disk that provides the appropriate factor for growth of
the organism
 Results:
[+] = Growth around XV disks
Haemophilus influenzae
= Growth around the V disks and no growth around X disk and light growth on the XV di sks
Haemophilus parainfluenzae
= Growth around the X disk and XV disks and no
growth around V disk
Haemophilus ducreyi

[-] = Growth over the entire surface of the agar indicates no requirement for either X or V factor

Haemophilus influenzae Haemophilus parainfluenzae Haemophilus ducreyi

Triple Sugar Iron Agar

 Purpose
o TSI is used to determine whether a gram negative rod ferments glucose and lactose or sucrose
and forms hydrogen sulfide(H2S)
o The test is used primarily to differentiate members of the Enterobacteriaceae family from other
gram negative rods
 Principle:
o pH indicator: Phenol Red to detect acid production
o H2S Production indicator: Ferrous Ammonium Sulfate
o Anaerobic decomposition through glucose utili zation wherein the entire butt turns acidic and
slant becomes acidic(yellow) when lactose and sucrose are being used up.
K= Alkaline (red)
A = Acid (yellow)

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 Result:
K= Alkaline (red) A = Acid (yellow)
K/K – Alkaline butt(K) and Alkaline Slant(K)
- Lactose, sucrose and glucose were not utilized
K/A – Alkaline slant (K) and Acid Butt(A)
- Only glucose is fermented
A/A – Acid Slant(A) and Acid butt(A)
- Glucose, sucrose, lactose fermenter

Gas Formation = bubbles and cracks observed on the agar indicating production of CO2 and
hydrogen gas (H2)
H2S production = blackening of agar
 All Enterobacteriaceae are enterogenic or gas producers except Shigella
 All Enterobacteriaceae are glucose fermenter
 K/K = Pseudomonas aeruginosa
 K/A, gas production, H2S production =Salmonella typhimurium
 A/A, gas production = Escherichia, Klebsiella and Enterobacter
 K/A = Shigella flexneri
 K/A and H2S production = Proteus mirabilis

Lysine Iron Agar(LIA)

 Purpose:
o To determine the ability of the organism (gram negative bacilli) to deaminate lysine,
decarboxylate lysine and produce hydrogen sulfide (H2S)
 Principle:
o Lysine iron agar contains lysine, peptones, a small amount of glucose, ferric ammonium citrate
and sodium thiosulfate
o The anaerobic butt becomes acidic when glucose is fermented.
o If the organism produces lysine decarboxylase, cadaverine is formed that neutralizes the organic
acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline
state(purple)
o If the decarboxylase is not produced, the butt remains acidic (yellow)

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o If Oxidative deamination of lysine occurs, a compound is formed that, in the presence of ferric
ammonium citrate and coenzyme, flavin mononucleotide, forms a burgundy color on the slant
o If deamination does not occur, the LIA slant remains purple
o pH indicator = Bromcresol purple
(A) = yellow
(K) = purple
o H2S indicator = Ferric Ammonium Citrate
SLANT = Detects organism capable of lysine deamination
(+) Red (R) = Proteus, Providencia, Morganella
(-) Purple (K)
BUTT = detects organism capable of lysine decarboxylation
(+) Purple (K)
(-) Yellow (A)

 Results:
K/K = (+)Lysine decarboxylation
(-) Lysine deamination
(-) glucose fermentation
Escherichia coli
K/K H2S+ = (+)Lysine decarboxylation
(-) Lysine deamination
(-) glucose fermentation
Salmonella, Arizona, Citrobacter, Edwardsiella

K/A = (-) Lysine decarboxylation

(-) Lysine deamination
(+) glucose fermentation
Shigella
R/A = (-) Lysine decarboxylation
(+) Lysine deamination
(+) glucose fermentation
Proteus, Providencia, Morganella

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SUMMARY

To identify bacteria, we must rely heavily on biochemical testing. The types of biochemical reactions each
organism undergoes act as a "thumbprint" for its identification. This is based on the following chain of logic:

Each different species of bacterium has a different molecule of DNA (i.e., DNA with a unique series of nucleotide
bases).
Since DNA codes for protein synthesis, then different species of bacteria must, by way of their unique DNA, be able
to synthesize different protein enzymes.
Enzymes catalyze all the various chemical reactions of which the organism is capable. This in turn means that
different species of bacteria must carry out different and unique sets of biochemical reactions.

REFERENCE

 Bailey & Scott's Diagnostic Microbiology 12e

 Introduction to Diagnostic Microbiology by Maria Dannessa Delost

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